CN1314707C - 2, 4, 6-trichlorophen artificial antigen, and its preparing method and use - Google Patents
2, 4, 6-trichlorophen artificial antigen, and its preparing method and use Download PDFInfo
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Abstract
The present invention relates to a 2, 4, 6-trichlorophenol artificial antigen, a preparation method thereof and a use thereof. The 2, 4, 6-trichlorophenol artificial antigen has a formula (I) showed on the right, wherein R is equal to BSA or OVA, NHBSA is bovine serum albumin, and NHBSA is egg albumin. The 2, 4, 6-trichlorophenol artificial antigen is synthesized by hapten 2, 4, 6-trichlorophenol oxygen acetic acid through an ester activation method or an acid anydride mixture method. The antigen can be used for preparing a 2, 4, 6-trichlorophenol antibody, the establishment of an indirect competing fluorescence immunity method of the 2, 4, 6-trichlorophenol or the detection of residual 2, 4, 6-trichlorophenol in a hydroenvironment. The present invention has the advantages that the practicability of the antibody is strong, and the stability of the antibody is good; the preparation method is simple and feasible, the cost is low, and the industrialization scale production of the preparation method is easy.
Description
Technical field
The present invention is a kind of 2,4, and 6-trichlorophenol artificial antigen, preparation method and its usage belong to the immunochemistry and the retention analysis biological technical field of small molecules organic pollutant.Relate to organic synthesis, immunochemistry, biological chemistry and physical and chemical testing technology etc.Be particularly related to environmental hormone class material 2,4, the preparation method of the synthetic and immune animal specific antibody of the design of 6-trichlorophenol haptens, artificial holoantigen, and set up can be applicable to detect in water body and the soil residual 2,4, the fluoroimmunoassay technology of 6-trichlorophenol.
Background technology
Environmental hormone is the hormone analogs in the environment, and it can disturb normal physiological metabolism, internal secretion and reproduction function by combining with hormone receptor, causes all negative biological effects.Over nearly 70 years, along with industrial expansion, a large amount of environmental hormones constantly discharges in processes such as the use of pharmacy, plastics Additive Production, weedicide and refuse treatment, and ecotope has been caused huge harm.The environmental hormone problem has become the third-largest environmental problem in the whole world after ozonosphere, terrestrial climate warm, becomes the heat subject in the Research of Environmental Sciences field.Screening that its main research range is an environmental hormone and investigation, mechanism of action, monitoring method, to harm of humans and animals etc.Kind of chemical was an environmental hormone class material surplus brainstrust had filtered out 70 at present, was used for making the daily coating of people, scouring agent, resin, plasticizer etc. more.That wherein lists environmental hormone class material in the phenolic compound in has alkylphenol, chlorophenol and a dihydroxyphenyl propane etc.[Jiang Baoxi, Li Wenlan. the present Research of Environmental Hormone and trend thereof. world environments, 2002,3:7-10]
Because environmental hormone has the characteristic of " low dosage; long-time " in environmental pollution, therefore, to environmental hormone class material 2,4, the 6-trichlorophenol directly monitor live toward difficulty relatively [Qi Wenqi, Sun Zongguang. the monitoring of organic micro-pollutant. Beijing: the .2002 of Chemical Industry Press].At present molecular weight is educated malicious pollutent such as environmental hormone class material less than 1000 daltonian small molecules classes, traditional analytical procedure mainly is a chromatography, comprises gas-chromatography, high performance liquid chromatography, application of gas chromatorgraphy/mass method (GC-MS, HPLC-MS) etc.But with determinand separation and Extraction and to remove coexistent impurity interferential pre-treatment process very complicated from sample, need to require high to laboratory and instrumentation degree in this alanysis method system through complicated sample pre-treatments such as extracting, purify, concentrate and derive.And during operational cost, the analysis cost costliness is unfavorable for promoting the use of, and is difficult to adapt to great amount of samples and the on-the-spot requirement that detects.Thereby seek the direction that a kind of quick, easy, sensitive and economical and practical analytical procedure becomes current research.
Immunoassay is the special reagent analysis method of a kind of form, and its starting point is that antibody becomes analytical reagent, utilizes antibody and antigenic specificity keying action to come selectivity identification and mensuration to can be used as antibody or antigenic determinand.Immunoassay is not subjected to the influence of surrounding environment and sample internal interference material, have highly sensitive, selectivity is good, stability is strong and the advantage of simple and convenient economy, the achievement that practical application at home and abroad obtains can not be underestimated, can carry out the detection of over one hundred micro substance, produced good social benefit and economic benefit [Wang Qinfu. immunology and immunological test experimental technique. Beijing: Chinese Medicine science and technology press, 1995].
As far back as eighties of last century the '30s, 2,4, the 6-trichlorophenol just is widely used in sterilant, sterilant and sanitas.It also is an intermediate of producing fibric acid sterilant and other chemical, is the by product of organic chlorine degradation in the water body.Also widespread use in printing and dyeing and paper industry, thereby its a large amount of use polluted soil and water source, causes environmental pollution.Because 2,4, the 6-trichlorophenol has carcinogenic, teratogenesis and mutagenic genotoxic potential, by China, European Union and Environmental Protection Agency list environment priority pollutants list [I.Intergrated-Risk-Infbrmation-System (IRIS electronic database) .Cincinnati.OH.USA in, 2001] [M.Fielding, D.Barcel ó, A.Helweg.S.Galassi, L.Tortensson, P.VanZonen, R.Wolter, G.Angeletti, Pesticides in Ground and Drinking Water, EuropeanCommission, Brussels, 1992].
2,4, the environmental influence that the 6-trichlorophenol brings and it have caused people's extensive concern to the genotoxic potential of organism.Provide its environmental health criteria according to the World Health Organization and USEPA, 2,4, the 6-trichlorophenol allows peak concentration in water body be 5 μ g/l, olfactory threshold concentration is at 0.001～0.0016mg/m in the air
3[WorldHealth Organization.Environmental health criteria for chlorophenols.Supplement.Draft, July 31,1986] [U.S.Environmental Protection Agency.Ambient water qualitycriteria for chlorinated phenols.EPA-440/5-80-032, NTIS Publ.No.PB81-117434, Office of Water Regulations and Standard (1980)]." water environment quality standard " that China carries out (GHZB1-1999) in regulation 2,4, the limit standard value of 6-trichlorophenol be 0.0012mg/L[" water environment quality standard " (GHZB1-1999), 2001].
Modern technology of preparing has obtained 2 of higher degree (＞99%), 4,6-trichlorophenol [chlornitrofen intermediate 2,4,6-trichlorophenol technology of preparing, fine-chemical intermediate, 1980,2:296], and relevant 2,4, the research of 6-trichlorophenol immunoassay has both at home and abroad and appears in the newspapers lessly, and people such as Roger Glave obtain anti-2 by immunize rabbit, 4,6-trichlorophenol antiserum(antisera) uses enzyme-linked immunosorbent assay (ELISA) to testing conditions optimization, but does not apply to [Roger Glave in the concrete environment measuring, Franciso Sanchez-Baeze, Franciso Camps, M.Pilar Marco, Indirect competitive immunoassay for trichlorophenol determination rationalevaluation of the competitor heterology effect, Analytica Chimica Acta, 2002,452:191-206] [Patricia Noguera, a Angel Maquieira, a Rosa Puchades
*A Ernesto Brunet, bMa Mar Carramolinob and Juan Carlos Rodn ' guez-Ubis.Development of anenzyme-linked immunosorbent assay for screening contamination by chlorophenols inenvironmental waters.J.Environ.Monit., 2002 (4): 442-448].And yet there are no report both at home and abroad for fluoroimmunoassay (FIA).The fluoroimmunoassay technology was firstly appeared the beginning of the forties, its ultimate principle is that fluorophor on specific antibody or the antigenic mark is made it to become specific reagent, with corresponding antigen or antibodies, form immune complex, detect fluorescent signal with fluorescence detector again.This method combines the specificity of immunological response and the susceptibility of fluorescence technique, has higher sensitivity and lower detectability than ELISA method.
The present invention utilizes 2,4 of preparation, and what 6-trichlorophenol artificial antigen immune animal obtained to have the height of tiring, specificity is good resists 2,4,6-trichlorophenol polyclonal antibody.It is selected that method is simple.
Summary of the invention
It is a kind of 2,4 that the object of the invention provides, 6-trichlorophenol artificial antigen.
It is a kind of above-mentioned 2,4 that purpose of the present invention also provides, the preparation method of 6-trichlorophenol artificial antigen.
Another object of the present invention provides a kind of above-mentioned 2,4, the purposes of 6-trichlorophenol artificial antigen.Specific antibody be can be used for preparing and water body and the residual environmental hormone class material 2,4 of soil, 6-trichlorophenol detected.
At first according to small molecules immunochemistry ultimate principle design synthesized micromolecule target analytes haptens, synthesized artificial holoantigen by appropriate means and carrier protein coupling, obtained small molecules analyte specific antibody with the holoantigen immune animal behind the purifying.Thereby the specificity immunology that utilizes antigen-antibody reacts trace small molecules target analytes in the qualitative, quantitative ground test sample, and its selectivity is decided by the specificity of immunological response, and the affinity of antibody is depended in its sensitivity.Therefore this technology can be used for environmental hormone class material 2,4 residual in rapid detection water body and the soil, 6-trichlorophenol.The key of this technology is the preparation of the synthetic and antibody of haptenic molecular designing, artificial antigen.
Of the present invention 2,4,6-trichlorophenol artificial antigen has following structural formula:
2,4, the molecular structural formula of 6-trichlorophenol is:
Its structural unit part is a phenyl ring, and another part is substituting group hydroxyl and the halogen on the phenyl ring.On the immunology angle, the phenyl ring space structure is relatively large, can be used as antigenic determinant.And phenolic hydroxyl group is compared with halogen, has higher chemically reactive, can be used as the arm that is connected with carrier proteins.Thereby when synthesizing haptens, keep phenyl, and the hydroxyl on the phenyl ring is modified, form and protein link coupled arm.Therefore this patent is when the design haptens, employing has active phenolic hydroxyl group and chloroacetate reaction, introduce the synthetic method of the different active side chain of structure and synthesize haptens, so both can keep 2,4,6-trichlorophenol haptens and 2,4,6-trichlorophenol similarity structurally can make haptens have suitable construction with carrier protein couplet again.
Synthetic 2,4 of the present invention, the haptenic molecular structure of 6-trichlorophenol is:
Chemical name: 2,4,6-trichlorophenoxyacetic acid, molecular weight are 255.5.
2,4,6-trichlorophenol haptens synthetic technical scheme is:
With basic solution dissolved chlorine acetate, with strong base solution dissolving 2,4, the 6-trichlorophenol adds little amount of catalyst polyoxyethylene glycol-6000, at 60～100 ℃ of following backflow stirring reaction 4～6h.Cool off then, promptly get thick product after the filtration, washing, drying, thick product gets finished product through recrystallization.Synthesis step is as follows:
A certain amount of Mono Chloro Acetic Acid is dissolved in 5～10ml water, stir and slowly add 2～3g powdered sodium carbonate down, to pH7～8, get a certain amount of 2,4, the 6-trichlorophenol adds an amount of water and 5ml 20～50% sodium hydroxide (or potassium hydroxide), make it to dissolve fully, regulate pH7～8, stir the above-mentioned sodium chloroacetate solution of adding down then, and adding little amount of catalyst polyoxyethylene glycol-6000 (0.01～0.05g), add and keep pH7～8,70～90 ℃ of reactions of continuation heating, 2～3h, the reaction solution of attention control simultaneously pH9～10 (regulating) with diluted alkaline, in 80～90 ℃ of insulation 2～5h, reaction finishes, and adds the small amount of activated decolouring, boils 5～10min, filtered while hot, filtrate is transferred pH1～2 with 1～2M hydrochloric acid, separates out white precipitate, filters out precipitation and washes with water, centrifugal drying gets crude product.With crude product acetic acid: water (1: 2～1: 4) heating for dissolving, decolouring after-filtration cooling recrystallization, finished product, productive rate is about 80%.
2,4,6-trichlorophenol artificial antigen synthetic technical scheme is:
(1) active ester method synthetic immunogen, step is as follows:
With amide group CONH is bridge, haptens is passed through active ester method, be connected respectively on the carrier protein (Protein), carboxyl on the haptens generates an intermediate with N-hydroxy-succinamide (NHS) reaction earlier, and then with protein on amino coupled, form haptens-proteinic conjugate.
Reaction formula is as follows:
The organic solvent that is dissolved with NHS and DCC dropwise added be dissolved with haptens 2,4, in the organic solvent of 6-trichlorophenoxyacetic acid, stirring at room reaction 6～12 hours, 4 ℃ are spent the night, the centrifugal supernatant liquor of telling.The Acibenzolar liquid on upper strata is joined in the protein soln that is dissolved in buffered soln 4 ℃ of reaction 2～6h.Described haptens, NHS, DCC, protein mol ratio are respectively 1: 2～4: 2～4: 0.01～0.05.
Reaction finishes, with the solution dialysis tubing of packing into, with distill water dialysis or the pH6.8～7.4 phosphate buffer soln PBS 5d that dialyses.The centrifugal supernatant liquor of telling in dialysis back promptly gets immunogen TCP-BSA, add ℃ freezing preservation down of equivalent glycerine-20, or-20 ℃ of freeze-drying is preserved.
(2) the synthetic coating antigen of mixed anhydride method, step is as follows:
Haptenic carbonyl with the isobutyl chlorocarbonate reaction, forms the mixed acid anhydride intermediate in the presence of n-Butyl Amine 99, with proteinic amino reaction, form haptens-protein conjugate again.
Reaction formula is as follows:
Get a certain amount of haptens 2,4 that is dissolved with, 6-trichlorophenoxyacetic acid organic solvent adds n-Butyl Amine 99 and isobutyl chlorocarbonate in turn, stirring at room reaction 2～4h.Above-mentioned reaction solution is dropwise gone into in the buffered soln dissolved protein soln 4 ℃ of reaction 1～3h.Be respectively 1: 2～4: 1.5～3: 0.01～0.05 by haptens, n-Butyl Amine 99, isobutyl chlorocarbonate, protein mol ratio.
Reaction finishes, with the solution dialysis tubing of packing into, with distill water dialysis or the pH6.8～7.4 phosphate buffer soln PBS 5d that dialyses.The centrifugal supernatant liquor that gets in dialysis back promptly gets coating antigen DCP-protein conjugate, after purifying through post, add ℃ preservation down of equivalent glycerine-20, or-20 ℃ of freeze-drying is preserved.
Described organic solvent is N, dinethylformamide DMF or methyl-sulphoxide DMSO.
Utilize 2,4 of above-mentioned haptens and protein synthesis, 6-trichlorophenol artificial antigen, its structural formula is as previously mentioned.
Utilize above-mentioned 2,4,6-trichlorophenol artificial antigen, 2,4 of immune animal preparation, 6-trichlorophenol specific antibody, it is can be with 2,4, the immunoglobulin IgG of 6-trichlorophenol generation specific immune response.
Above-mentioned 2,4,6-trichlorophenol specific antibody is used for detecting water body and soil 2,4, the residual quantity of 6-trichlorophenol.
The present invention is by design Synthetic 2,4,6-trichlorophenol haptens and artificial antigen, produce specific antibody through immune animal,, and introduce marker and amplify this reaction of demonstration based on the antigen and antibody specific immunological response, can be used in the actual sample 2,4, the mensuration of 6-trichlorophenol residual quantity.Highly sensitive, the high specificity of this method, sample pre-treatments is simple, is convenient to carry out field monitoring.
Characteristic feature of an invention
The invention provides a kind of 2,4, the haptenic synthetic route of 6-trichlorophenol.The invention provides a kind of 2,4, the synthetic method of 6-trichlorophenol artificial antigen.The present invention also provide a kind of have anti-2,4 than high specific, the simple and feasible preparation method of 6-trichlorophenol antibody.The invention provides a kind ofly 2,4, the fluorescence immunoassay of 6-trichlorophenol antibody detects and analytical technology.
The present invention also has the following advantages and positively effect:
(1) antibody is practical: 2,4, and 6-trichlorophenol antibody production techniques has important use value and practical significance.Good, the high antibody of tiring of specificity of preparation is that the basis can be used for sample determination with the antigen-antibody immunological response.This antibody and preparation method thereof is immunoassay 2,4, and the 6-trichlorophenol provides safeguard, and also is environmental hormone 2,4, and the development of 6-trichlorophenol quick detection kit has solved technological difficulties.
(2) antibody good stability: 2,4 of this method preparation, 6-trichlorophenol antibody have stability preferably.
(3) antibody production techniques is simple and feasible: the whole process of preparation of antibody need not special plant and instrument, and is with low cost, commercial scale production easily.
Other purpose, characteristics and advantage of the present invention can be confirmed from the description of optimization experiment scheme.
Specific embodiment
Embodiment 12, and 4, the haptenic synthetic and evaluation of 6-trichlorophenol
12,4, the 6-trichlorophenol is haptenic synthetic
4.5g (47.6mmol) Mono Chloro Acetic Acid is dissolved in the 7.5ml water, slowly adds 2.55g powdered sodium carbonate, pH value of solution 7.5 under stirring.Get 6.7g (38.1mmol) 2,4,6-trichlorophenol and 10ml water, 5ml 25% sodium hydroxide makes it to dissolve fully, pH value of solution 8, stir the above-mentioned sodium chloroacetate solution of adding down then, add maintenance pH7～8 (regulating), add 0.08g catalyst polyethylene glycol-6000 with diluted alkaline, continue reacting by heating 2h, note control reaction solution pH9～10 (regulating with diluted alkaline) simultaneously, in 80～90 ℃ of insulation 3h, reaction finishes, add the small amount of activated decolouring, boil 10min, filtered while hot, filtrate is transferred pH1 with hydrochloric acid, separate out white precipitate, filter out the precipitation and wash with water, centrifugal drying, crude product.With crude product acetic acid: water (1: 2) heating for dissolving, decolouring after-filtration cooling recrystallization, finished product, productive rate is about 81%.
22,4, the haptenic evaluation of 6-trichlorophenol
Get above-mentioned synthetic product through infrared spectra IR and nuclear magnetic resonance spectrum
1H-NMR measures molecular structure.IR (KBr compressing tablet) cm
-1: 3425 (w, OH), 1765 (w, C=O), 1697 (s, sub-COOH), 1423 (s, CH
2), 1251 (m, C-O), 1079 (m, C-O-C), 930 (m, COOH).
1H-NMR(CDCl
3)：δ(ppm)：7.35(2H，ArH)，4.47(2H，CH
2)。From above as can be known analysis integrated, institute's synthetic product is a target compound.
Embodiment 22, and 4, the synthetic and evaluation of 6-trichlorophenol artificial antigen
With haptens 2,4,6-trichlorophenol phenoxy acetic acid adopts active ester method and mixed anhydride method to be connected on BSA and the OVA respectively, synthetic artificial antigen (immunogen and coating antigen), and concrete grammar is:
2.1 it is immunogenic synthetic
Take by weighing 0.2mmol 51mg haptens 2,4,6-trichlorophenol phenoxy acetic acid is dissolved among the 200 μ l DMF, and 19.4mg NHS and 34.2mg DCC are dissolved in 300 μ l DMF, dropwise add in the haptens solution, and stirring at room reaction 8h, 4 ℃ are spent the night, the centrifugal supernatant liquor of telling.With the Acibenzolar liquid on upper strata join 60mg BSA be dissolved in the 5ml carbonate buffer solution (0.05M, pH9.6), 4 ℃ of reaction 4h.Reaction finishes, and with the solution dialysis tubing of packing into, uses distill water dialysis 2d, the pH7.0 phosphate buffer soln PBS 3d that dialyses.The centrifugal supernatant liquor of telling in dialysis back promptly gets immunogen TCP-BSA, adds ℃ freezing preservation down of equivalent glycerine-20.
2.2 coating antigen is synthetic
Take by weighing 0.2mmol 51mg haptens 2,4, the 6-trichlorophenoxyacetic acid is dissolved among the 200 μ l DMF, adds 20 μ l n-Butyl Amine 99s and 20 μ l isobutyl chlorocarbonates in turn, stirring at room reaction 1h.Other get 40mg OVA be dissolved in 3ml yellow soda ash buffered soln (0.05M pH9.6), dropwise adds above-mentioned reaction solution 100 μ l, 4 ℃ the reaction 2h.Reaction finishes, and with the solution dialysis tubing of packing into, uses distill water dialysis 2d, the pH7.0 PBS 3d that dialyses.The centrifugal supernatant liquor that gets in dialysis back promptly gets coating antigen TCP-OVA, adds equivalent glycerine and preserves down for 4 ℃.
2.3 the evaluation of artificial antigen
Adopt the long ultraviolet spectrophotometer of all-wave that haptens, carrier proteins and conjugate are scanned, determine according to absorption peak whether coupling is successful.The result shows that haptens has maximum absorption band at the 324nm place, PROTEIN B SA and OVA have maximum absorption band at the 280nm place, and conjugate immunogen charateristic avsorption band is 275nm and 312nm, the coating antigen charateristic avsorption band is 276nm and 308nm, and displacement has more all taken place for the absorption peak of conjugate and protein, haptens.Conjugate possesses has haptenic absorption feature to illustrate that also the synthetic of artificial antigen is successful.According to haptens-binding substances the absorption in the uv-absorbing zone approximate simply adding of floating preteins and haptenic light absorption value and characteristics, by the three at the light absorption value of 280nm by following formula calculations incorporated ratio:
Binding ratio=[ε
280 antigens-ε
280 protein]/ε
280 haptens
Through calculating, binding ratio is as follows: immunogen TCP-BSA binding ratio is that 17: 1, coating antigen TCP-OVA binding ratio are 11: 1.This paper synthetic 2,4,6-trichlorophenol artificial antigen all within the scope that document is advised, has proved institute's synthetic 2,4 in conjunction with ratio, 6-trichlorophenol artificial antigen is more satisfactory.
Embodiment 32, and 4, the preparation and the purifying of 6-trichlorophenol antibody
3.1 sero-fast preparation
Choose two every group of adult male New Zealand rabbits (numbering 1 and II), the about 2～2.5Kg of body weight raises in the standard test Animal House, observes week back immunity (being raised by agricultural college of Shanghai Communications University Animal House).
Immunogen is slowly thawed, rare with 0.9% physiological saline (or phosphate buffer soln) to 1mg/ml, suck in the 5ml sterile syringe by immunizing dose, first immunisation adds equivalent Freund's complete adjuvant (CFA), with another 5ml syringe of fluorinated ethylene propylene plastic cement pipe coupling, fully emulsified to being pushed into, form water-in-oil (W/O) state, press the immunization protocol injecting immune.
First immunisation adopts by equivalent Freund's complete adjuvant emulsive immunogen, in the immunity of rabbit back intracutaneous multi-point injection.Head exempts from around the interval, back, and every using in two weeks by Freund's incomplete adjuvant emulsive immunogen, in rabbit back, the subcutaneous multiple spot of neck and shank injection booster immunization, immunizing dose is that per kilogram is exempted from body weight 2mg later on.From for the third time, back 7 days of each immunity is adopted a small amount of blood in rabbit ear edge vein, and separation of serum is measured antibody titer.When reach necessarily tire after, adopt whole blood.Rabbit carotid artery blood taking method is adopted in this experiment, every rabbit can be taken a blood sample about about 60ml, after the blood sampling, is collected in the triangular pyramidal bottle of 100ml, after treating blood coagulation, separate with centrifuge tube with the clot of syringe needle, place 37 ℃ of incubator 30min, be put in 4 ℃ of refrigerator 3～4h again the triangular flask edge, after blood clot retraction, with suction pipe serum is sucked in the centrifuge tube,, isolate serum with the centrifugal 15min of 4000rpm.In-20 ℃ of preservations.The I rabbit gets serum 20ml, and the II rabbit gets serum 25ml.
3.2 antibody titer is measured
With immunogen by the immunization protocol immunity of formulating two rabbits.From booster immunization for the third time, adopted a small amount of blood in rabbit ear edge vein on the 7th day in each immunity back, serum is after suitable dilution, with agar double diffusion test and fluorescent immune method mensuration antibody titer.After treating the 4th immunity, rabbit has obtained higher tiring, and tiring that agar double immunodiffusion method records is respectively 1: 32 and 1: 64, and fluorescent immune method records to tire and was respectively 1: 25600 and 1: 51200 (antiserum(antisera) fluorescent value/negative control fluorescent value＞2).
3.3 purifying antibody and evaluation
Purifying antibody adopts sad-two step of saturated ammonium sulphate salting-out process, through sephadex column desalination and the anti-phase adsorption and purification serum of DEAE-cellulose post, after the antibody purification freeze-drying in-20 ℃ of preservations.Sad can be under the condition of slant acidity with serum in protein except that immunoglobulin IgG all precipitate, have only IgG in the supernatant liquor.
Embodiment 42, and 4, the foundation of 6-trichlorophenol indirect competition fluorescence immunoassay method (FIA)
4.1 the principle of fluorescence immunoassay method
The indirect competition fluoroimmunoassay is that the mixture with micromolecular chlorophenols environmental hormone and macromolecular carrier coupling preparation is adsorbed on the solid phase carrier (96 hole luciferase target) as envelope antigen, make solid phase antigen, add chlorophenol to be measured then, chlorophenol in the solid phase antigen and chlorophenol to be measured and the antibody association reaction that is at war with, if chlorophenol content to be measured is many more, it is just few more to be bonded to the antibody of insolubilized antibody on former, otherwise the antibody that is combined in solid phase antigen is then many, that reaction back adds is fluorescently-labeled two anti-(can only with the antibodies that is combined on the solid phase antigen), the free unreacted determinand of last flush away, 2,4,6-trichlorophenol antibody and two resists, and places reading on the fluorescence microplate reader.When one timing of antibody amount, the chlorophenol amount to be measured that adds is many more, just few more with solid phase antigen bonded antibody, fluorescence intensity just weakens, and inhibiting rate increases, otherwise, then fluorescence intensity increases, inhibiting rate increases, and therefore can extrapolate the concentration of chlorophenol to be measured according to the chlorophenol typical curve of known quantity and the inhibiting rate of testing sample.
4.2 determining of antigen and antibody best effort concentration
Respectively antigen and antibody working concentration in the indirect competition FIA method are screened and assert with square formation test.Under same coating buffer concentration, along with the dilution of antibody concentration, the fluorescence intensity of gained is decline, and under same antibody concentration, along with the decline of coating buffer concentration, the fluorescence intensity of gained also is decline equally.To suppress highly sensitive, the test combinations of good linearity is a best effort concentration.The result judges with among each the hole fluorescent value F that reads at fluorescence microplate reader, and is bigger with Δ F reading, and the antigen-antibody consumption less be the working concentration of antigen-antibody.Selecting envelope antigen concentration is 2 μ g/ml, and antibody concentration is that 3.5 μ g/ml are best effort concentration,
4.3 the drafting of typical curve
According to the square formation test-results, minimum with blank absorption value, fluorescence intensity is better, and 2,4, the 6-trichlorophenol is to antigen-antibody binding reaction inhibiting rate height, and typical curve is set up in the test conditions combination that linear relationship is good.Its step is as follows:
(1) bag quilt: with 0.05mol/L pH9.6 carbonate buffer solution envelope antigen is diluted to working concentration, 100 μ L/ holes add to the luciferase target, and 4 ℃ are spent the night.
(2) sealing: will wrap by good plate and take out, and treat that it will return to room temperature, PBST washing 3 times.Add the 1%OVA sealing, 150 μ L/ holes, 37 ℃ of incubation 0.5h.
(3) application of sample: will seal good plate and take out, and treat that it will return to room temperature, PBST washing 3 times.Every hole adds 50 μ L successively through 2,4 of the series concentration of PBST dilution, and accurate solution in 6-trichlorophenol border and 50 μ L are through the antibody of PBST dilution, and control wells adds 50 μ L PBST, 37 ℃ of incubation 1h.
(4) two anti-reactions: the plate that incubation is good takes out, and treats that it returns to room temperature, PBST washing 3 times.Every hole adds 100 μ LPBST dilution goat-anti rabbit FITC-IgG (1: 1000), 37 ℃ of incubation 1h.
(5) measure: the plate that incubation is good takes out, PBST washing 5 times.Read fluorescence intensity level F with fluorescence microplate reader, λ is made as 528nm.
(6) according to inhibiting rate to 2,4,6-trichlorophenol concentration mapping obtains 2,4, the regression equation of the typical curve of 6-trichlorophenol carries out correlation analysis, and calculates IC
50And IC
20
To 2,4, map by the logarithm of 6-trichlorophenol with inhibiting rate I for the typical curve of FIA method.The calculation formula of inhibiting rate is as follows:
The I-inhibiting rate, F
Max-light absorption value when not adding the chlorophenol sample, F
SFluorescent value during-Jia chlorophenol sample, F
MinThe fluorescent value in-blank hole (or the fluorescent value in negative control value hole)
Can get 2,4 by following formula, the inhibiting rate of each concentration of 6-trichlorophenol, mapping.The result shows that when 2,4, the concentration of 6-trichlorophenol is in 1～100 μ g/L scope, inhibiting rate I and 2,4, the logarithm of 6-trichlorophenol is the linear relationship of expressing, its equation of linear regression is I=27.16x+20.42, correlation coefficient r=0.9965, concentration in the inhibition (is 50% expression with inhibiting rate) IC
50=12.3 μ g/L, lowest detectable limit (is 20% expression with inhibiting rate) IC
20=0.96 μ g/L.
4.4 the specificity of antibody
The specificity of antibody promptly is meant the comparison of it and homospecificity antigen bonded ability and antigen-analogues ability, often with the standard of cross reacting rate as evaluation.Cross reaction is more little, and then the specificity of antibody is good more.Choose and 2,4, the compound Pentachlorophenol, 2 of 6-trichlorophenol structural similitude, 4-two chlorophenols, 2-chlorophenol, 4-chlorophenol etc. are with 2,4, and the 6-trichlorophenol is tested by FIA test carrying out cross reaction under the same conditions, thereby measures 2,4, the specificity of 6-trichlorophenol antibody.According to 2,4, the inhibition curve of 6-trichlorophenol analogue is obtained concentration IC in the inhibition of different chlorophenols
50, calculate cross reacting rate according to following formula:
2,4,6-trichlorophenol antibody is to Pentachlorophenol, 2, and the cross reacting rate of 4-two chlorophenols, 2-chlorophenol, 4-chlorophenol etc. is respectively 0.6%, 13.8%, 6.2%, 4.9%.Thereby as can be known, the specificity of three kinds of prepared antibody is stronger.
The FIA rapid determination of 5 samples
5.1 sample pretreatment
(1) water sample: get river, tap water and lake water behind sedimentation and filtration, regulate pH6.8 with hydrochloric acid, add 2,4 of different concns respectively, 6-trichlorophenol standardized solution carries out mark-on with indirect competition FIA method and reclaims mensuration, calculate recovery rate.
(2) pedotheque: the soil 10g that takes by weighing local different zones collection respectively sieves through 100 orders, places the 100mL triangular flask, spends the night with 30mL 10% alcohol dipping, after filtering, concentrating, is settled to 10mL with PBS, uses for detecting.Pipette through the sample solution after repeatedly measuring, add 2,4 of different concns respectively, 6-trichlorophenol standardized solution carries out mark-on with indirect competition FIA method and reclaims mensuration, calculate recovery rate.
5.2 the mensuration of sample
The sample determination method is identical with the operation of typical curve, with through bag quilt and sealing good the every hole of plate add the sample liquid 50 μ l of serial known interpolation concentration, control wells adds 100 μ l antibody, behind 37 ℃ of incubation 1h, washing, surplus back step is the same.By analysis as can be known, this method is used for water sample 2,4, and the mensuration that the 6-trichlorophenol is residual, the rate of recovery are between 96.0～116.4%, and variation coefficient scope is between 2.45～9.64%.Along with the reduction of sample addition, the rate of recovery is on a declining curve substantially.This method is used for soil 2,4, and the mensuration that the 6-trichlorophenol is residual, the rate of recovery are between 91.7～109.6%, and variation coefficient scope is between 2.63～11.82%.Along with the reduction of sample addition, the rate of recovery is on a declining curve substantially.
Embodiment 5 envelope antigens and antibody stability experiment
Fast detection method has very high requirement for artificial antigen and antibody degree of stability at normal temperatures, so the present invention has carried out confirmatory experiment.Luciferase target after bag is by, sealing, washing directly is inverted or with the preservative film sealing after be inverted in 4 ℃ of refrigerators and preserve the influence that the results of regular determination period of storage is analyzed FIA.Synthetic artificial antigen of the present invention has stability preferably, can preserve 6 months unchangeability under 4 ℃ of environment, can preserve 12 months at least in-20 ℃ of refrigerators.
The stability experiment result of lyophilized antibodies shows that its stability can keep more than 12 months at least-20 ℃ of preservations; 4 ℃ of preservations, its stability can keep more than 6 months at least; 0～25 ℃ of room temperature preservation, its stability can keep more than 2 months at least.Can satisfy the rapid detection requirement.
Although with reference to being considered to embodiment preferred at present, the present invention is not subjected to the restriction of disclosed invention.On the contrary, in the spirit and scope of suffered claim, the scheme that the present invention includes various changes and be equal to.The scope of following claim gives to explain the most widely, so that comprise all these changes and equivalent structure and function.
Claims (7)
1. one kind 2,4,6-trichlorophenol artificial antigen, it has following structural formula:
Wherein, R=BSA or OVA, BSA are bovine serum albumins, and OVA is an ovalbumin.
2. as claimed in claim 1 a kind of 2,4, the preparation method of 6-trichlorophenol artificial antigen is characterized in that adopting following two kinds of methods to obtain respectively:
(1) active ester method synthetic immunogen, step is as follows:
In under organic solvent and room temperature, haptens 2,4,6-trichlorophenol phenoxy acetic acid, N-hydroxy-succinamide NHS and N, N-dicyclohexylcarbodiimide reaction 6～12h, 4 ℃ are incubated 4～10 hours, the centrifugal supernatant liquor of telling; The Acibenzolar liquid on upper strata is joined in the carbonate buffer solution of pH9.6 of bovine serum albumin 4 ℃ of reaction 2～6h; Dialysed 5 days with distill water dialysis or pH6.8～7.4 phosphate buffer solns then; The centrifugal supernatant liquor of telling promptly gets immunogen; Described 2,4,6-trichlorophenol phenoxy acetic acid, N-hydroxy-succinamide, N, the mol ratio of N-dicyclohexylcarbodiimide and bovine serum albumin is 1: 2～4: 2～4: 0.01～0.05, described organic solvent is N, dinethylformamide
The immunogen structural formula is:
Or
(2) the synthetic coating antigen of mixed anhydride method, step is as follows:
In organic solvent, haptens 2,4,6-trichlorophenoxyacetic acid, n-Butyl Amine 99 and isobutyl chlorocarbonate stirring at room reaction 2～4h; Yellow soda ash buffered soln and above-mentioned reaction solution that other gets the pH9.6 that contains ovalbumin react 1～3h at 4 ℃; Dialysed 5 days with distill water dialysis or pH6.8～7.4 phosphate buffer soln PBS then; The centrifugal supernatant liquor that gets promptly gets coating antigen; Described haptens 2,4, the mol ratio of 6-trichlorophenoxyacetic acid, n-Butyl Amine 99, isobutyl chlorocarbonate and ovalbumin are 1: 2～4: 1.5～3: 0.01～0.05;
Wherein, the coating antigen structural formula is:
Wherein, BSA and OVA are according to claim 1.
3. as claimed in claim 2 a kind of 2,4, the preparation method of 6-trichlorophenol artificial antigen is characterized in that organic solvent is N, dinethylformamide or methyl-sulphoxide.
4. as claimed in claim 2 a kind of 2,4, the preparation method of 6-trichlorophenol artificial antigen it is characterized in that described immunogen or coating antigen ℃ preserve down at equivalent glycerine-20, or-20 ℃ of freeze-drying is preserved.
5. as claimed in claim 2 a kind of 2,4, the preparation method of 6-trichlorophenol artificial antigen is characterized in that (1) described haptens 2,4, and 6-trichlorophenol phenoxy acetic acid is made by following method:
In the presence of aqueous solution catalyst neutralisation polyoxyethylene glycol-6000, contain 2,4,20～50% sodium hydroxide of 6-trichlorophenol or the aqueous solution and the sodium chloroacetate solution of potassium, 70～90 ℃ of reacting by heating 2～3h regulate control reaction solution pH9～10 with diluted alkaline, in 80～90 ℃ of insulation 2～5h, reaction finishes, add the small amount of activated decolouring, boil 5～10min, filtered while hot, filtrate is transferred pH1～2 with 1～2M hydrochloric acid, separate out white precipitate, filter out precipitation and wash centrifugal drying with water, get crude product, with crude product acetic acid: water (1: 2～1: 4) heating for dissolving, decolouring after-filtration cooling recrystallization, finished product, productive rate is about 80%
The hapten molecule structure is:
One kind as claimed in claim 12,4, the purposes of 6-trichlorophenol artificial antigen is characterized in that being used to prepare 2,4,6-trichlorophenol antibody, 2,4, residual 2,4 in water body and the soil in the reagent of the indirect competition fluorescence immunoassay method of 6-trichlorophenol, the environment, the detection of 6-trichlorophenol.
7. as claimed in claim 6 a kind of 2,4, the purposes of 6-trichlorine artificial antigen is characterized in that described preparation 2,4,6-trichlorophenol antibody, and step is as follows:
(1) two every group of adult male New Zealand rabbits are chosen in experiment, about 2～the 2.5Kg of body weight, raise in the standard test Animal House, with 0.9% physiological saline or phosphate buffered saline buffer that immunogen is rare to 1mg/ml, first immunisation adds the equivalent Freund's complete adjuvant, fully emulsified, form the water-in-oil state, injecting immune; Head exempts from around the interval, back, and every using in two weeks by Freund's incomplete adjuvant emulsive immunogen, in rabbit back, the subcutaneous multiple spot of neck and shank injection booster immunization, immunizing dose is that per kilogram is exempted from body weight 2mg later on; From for the third time, back 7 days of each immunity is adopted a small amount of blood in rabbit ear edge vein, and separation of serum is measured antibody titer; When reach necessarily tire after, carotid artery is got blood, isolates serum, in-20 ℃ of preservations;
(2) purifying antibody adopts sad-two step of saturated ammonium sulphate salting-out process, obtains the higher immunoglobulin IgG of purity through sephadex column desalination and the anti-phase adsorption and purification serum of DEAE-cellulose post.
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| CN102062775A (en) * | 2010-12-10 | 2011-05-18 | 福州大北农生物技术有限公司 | Immunodetection kit for detecting porcine reproductive and respiratory syndrome virus and application thereof |
| CN105111307A (en) * | 2015-08-07 | 2015-12-02 | 上海交通大学 | Preparation and use of tris(2,3-dibromopropyl)isocyanurate coated antigen |
| CN105037525A (en) * | 2015-08-07 | 2015-11-11 | 上海交通大学 | Tris(2,3-dibromopropyl) isocyanurate (TBC) artificial immunogen TBC-BSA (bovine serum albumin) and preparation and usage thereof |
| CN105198985A (en) * | 2015-08-20 | 2015-12-30 | 上海交通大学 | Preparation and application of dimethyl phthalate artificial coating antigen (DMP-OVA) |
| CN105085668A (en) * | 2015-08-20 | 2015-11-25 | 上海交通大学 | Artificial coating antigen DEHP (diethylhexyl phthalate)-OVA of DEHP as well as preparation method and application thereof |
| CN105085662A (en) * | 2015-08-20 | 2015-11-25 | 上海交通大学 | Preparation method and application of artificial immunogen DBP-BSA of dibutyl phthalate |
| CN105198984A (en) * | 2015-08-20 | 2015-12-30 | 上海交通大学 | Dimethyl phthalate artificial immunogen and preparation and application thereof |
| CN105085663A (en) * | 2015-08-20 | 2015-11-25 | 上海交通大学 | Diethyl phthalate artificial immunogen, and preparation and application thereof |
| CN105085667A (en) * | 2015-08-20 | 2015-11-25 | 上海交通大学 | Diethyl phthalate artificial coating antigen as well as preparation and application thereof |
| CN107058240B (en) * | 2017-03-20 | 2019-08-13 | 江南大学 | One strain of hybridoma strain AB1 and its 2,4,5 trichlorophenoxyacetic acid monoclonal antibody of generation |
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